We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

모든 절차는 안과 및 비전 연구에서 동물의 사용을위한 비전 및 안과 문에 연구를위한 협회 부여 본 연구에서 다음과 국립 대만 대학 병원의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. 격리, 차 문화의 제조 및 소 CECs의 면역 염색 <ol><li> 로컬 도살장에서 신선한 소 눈을 획득. </li><li> 3 분 동안 10 %의 v / w 포비돈 요오드 용액에 눈 소독. 인산염 완충 식염수 (PBS) 솔루션을 씻으십시오. </li><li> 무균 조건에서 메스와 가위로 각막 버튼을 수확. 해부 현미경 집게로 껍질 데스 메막 (이 연구에서 그주의, 소 눈은 로컬 도살장에 의해 적출 된, 따라서 첫 번째 연구 절차는 실험실에서 preenucleated 눈의 소독이었다). </li><li> 시도 1 ㎖에 데스 메 막을 품어30 분 동안 37 ° C에서 psin. 5 분 112 XG에서 원심 분리하여 소 CECs를 수집합니다. 보충 호르몬 상피 매체 (SHEM) HEPES 완충 둘 베코 변형 이글 배지와 5 % 소 태아 혈청이 보충 된 햄 F12 배지, 0.5 % 디메틸 술폭 시드, 인간 표피 2 ng를 / ㎖의 동일한 양을 함유하는 1 ml의 세포를 재현 탁 성장 인자, 5 밀리그램 / 인슐린 ㎖, 5 밀리그램 / 트랜스페린 ㎖, 5 NG / 셀레늄 ㎖, 콜레라 독소의 1 nmol의 / l, 50 밀리그램 / 젠타 마이신 ㎖, 1.25 밀리그램 / 암포 테리 신 B의 용액 </li><li> 6 형상 접시에 세포 (안구 당 ​​약 1 × 105 세포)를 시드. 셈 문화를. 공기 중 5 % CO 2 분위기하에 37 ℃에서 인큐베이션 접시. 3 일마다 배지를 변경합니다. </li><li> 세포가 합류점에 도달하면, PBS로 씻어, 5 분 동안 37 ° C에서 트립신 1 ㎖ 그들을 부화. 5 분 112 XG에서 원심 분리를 수집합니다. 셈 1 ㎖의 세포 펠렛을 다시 일시 중지합니다. <ol&gt; <li> 혈구의 세포를 계산합니다. A A 24 웰 플레이트에 웰 당 1 × 104의 밀도, 문화 셈의 셀에서 커버 슬라이드에 세포를 시드. 타틴의 EnMT 억제 효과를 조사하는 경우, 타틴의 μM이 배양 배지에 첨가 10 SHEM에서 세포를 배양하고, 3 일마다 배양액을 변경합니다. </li></ol></li><li> 실온에서 30 분 동안 4 % 파라 포름 알데하이드 250 ㎕를, pH가 7.4으로 지시 된 시점에서 세포를 고정한다. 5 분 동안 0.5 % 트리톤 X-100 250 ㎕를 함께 Permeabilize 하시려면. 30 분 동안 10 %의 소 혈청 알부민으로 차단. </li><li> 밤새 4 ℃에서 활성 베타 - 카테닌 (ABC), 달팽이와 민달팽이에 대한 일차 항체와 세포를 품어. 200 : 사용 된 일차 항체의 희석은 1입니다. 항체는 10 mM의 PBS, 1 %의 v / w 소 혈청 알부민 및 0.09 % w / V의 아 지드 화 나트륨으로 구성된 항체 희석제로 희석된다. </li><li> 15 분 동안 PBS로 두 번 세포를 세척하고, 품어1 시간 동안 실온에서 (항체 희석액 100 1) 알렉사 형석 접합 이차 항체 m. </li><li> 4 &#39;, 6-diamidino-2-페닐 인돌 2 μg의 / ml의 세포를 피복하여 세포 핵 Counterstain과 15 분 동안 PBS로 두 번 세포를 씻어 장착 용액을 안티 페이드로 마운트. </li><li> 20X 목적 배율 레이저 스캐닝 공 초점 현미경을 사용하여 405 및 488 nm의의 여기 파장에서 면역 형광 이미지를 얻습니다. </li></ol> 2. 쥐 각막 내피 Cryoinjury 모델 및 내원시 주입 <ol><li> 2 % 자일 라진 (5.6 ㎎ / ㎏) 및 tiletamine 플러스 zolazepam (/ kg 18 mg)을 근육 주사이 12 주령의 수컷 흰쥐를 마취. 가볍게 피부 경련의 부재하에 적절한 마취를 확인하기 위하여 동물의 피부를 끼우는. </li><li> 눈의 통증과 깜짝 반사를 최소화하기 위해 각 쥐의 오른쪽 눈에 0.5 % proparacaine 염산염 한 방울을 주입. 주입왼쪽 눈에 테트라 사이클린 연고는 각막 건조를 방지합니다. </li><li> 액체 질소 스테인레스 스틸 프로브 (직경 = 3 mm)를 냉각. 30 초간 오른쪽 눈의 중심 각막 스테인리스 프로브를 적용한다. 자주 각막 건조를 방지하는 절차 중 우측 눈에 PBS를 주입. </li><li> 즉시 cryoinjury 후 0.1 % 아트로핀 및 0.3 % 황산 젠타 마이신을 주입 매일 한번 섬모 경련으로 인한 안구 통증을 완화하고, 감염을 방지 할 수있다. <ol><li> 시술 후, 래트를 가열 램프를 이용하여 보온, 그들은 모터 제어를 회복 할 때까지 마취 회복 후에도 15 분마다 관찰. 또한 회복 기간 동안 각막의 건조를 방지하기 위해 우안에 테트라 사이클린 연고를 적용한다. </li></ol></li><li> 3 일 연속 각막 cryoinjury를 반복합니다. </li><li> 전술 한 바와 같이 쥐 눈의 전방 챔버로 타틴 또는 bFGF를 전달 들어, 래트를 마취. 한 방울을 주입오른쪽 눈에 0.5 % proparacaine 염산염의 눈의 통증과 깜짝 반사를 최소화합니다. </li><li> 멸균 PBS와 안구 표면을 관개. 홍채에 위의 평행 한 평면에 paralimbal 맑은 각막에 1 ML의 주사기에 부착 된 30 G 바늘을 삽입하여 운영 현미경으로 전방 천자를 수행합니다. </li><li> 바늘이 최대 베벨 켜고, 약간 약간의 방수를 배출하고 안압을 줄이기 위해 각막 상처를 우울. 에 각막 약 0.02 mL의 주입. 조심스럽게 바늘을 철수하는 동안면 끝이 바늘 관을 압축합니다. </li><li> 운영 현미경 지시 된 시점에서, 외부 눈 사진. </li></ol> 3. 수확 쥐 각막 버튼과 면역 염색 <ol><li> 래트를 안락사하려면 안락사 챔버에 배치 분당 챔버 부피 10-30 %의 충전 속도로 100 % CO 2 달이다. 에 대한 CO 2 주입 유지호흡의 부족 및 머 금고 눈 색깔 후 추가 분. </li><li> 날카로운 블레이드와 윤부에서 쥐의 눈을 침투. 윤부를 따라 각막 가위로 각막을 잘라. 슬라이드에 각막을 평평하게. 각막이 꼬고 경우 추가 반경 방향 절개를합니다. </li><li> 실온에서 30 분 동안 4 % 파라 포름 알데하이드 250 ㎕를, pH가 7.4으로 각막을 수정. 5 분 동안 0.5 % 트리톤 X-100 250 ㎕를 Permeabilize 하시려면로, 30 분 동안 10 %의 소 혈청 알부민으로 차단. </li><li> (1)의 희석 4 ° C에서 하룻밤 ABC에 대한 일차 항체로 각막을 품어 : (200) 항체 희석액에. 15 분 동안 PBS로 두 번 씻어 각막 및 이차 항체로 배양 한 1 시간 동안 실온에서 (1 항체 희석제 100). 셀 2 μg의 4 / ㎖ &#39;, 6-diamidino -2- 페닐 인돌로 세포를 피복함으로써 핵 및 15 분 동안 PBS로 두 번 세포를 씻어 Counterstain과. </li><li> 설치 솔루션을 방지 페이딩의 각막을 탑재합니다. 산부인과20X 대물 확대 한 레이저 스캐닝 공 초점 현미경으로 면역 (405)의 여기 파장 및 561 nm의 이미지를 주석 박. </li></ol>

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The authors have no competing financial interests to declare.

CECs는 세포 증식 동안 EnMT를 받아야하는 그들의 성향 알려져있다. 치료 목적 EnMT 처리를 억제하기위한 전략을 개발하려면 EnMT기구의 완전한 이해가 필요하다. 우리는 EnMT, 체외 배양 모델 쥐의 각막 내피 세포의 cryoinjury 모델, 즉 소 CEC를 조사하기 위해이 모델을 설명했다. 우리의 결과는 두 모델의 EnMT 과정을 보여 주었다. 또한, 타틴의 EnMT 억제 효과는이 두 모델은 동일한 메커니즘을 공유 할 것을 제안, 두 모델에서 재현되었다. 체외 배양 모델의 소 CEC는 조작의 용이성을 제공합니다. 또한, 이전의 연구 (17, 18)에 사용되는 체외 배양 모델에서 인간 영장류 또는 CEC 비해 소의 눈을 더 크고 더 확보하기 쉽게하고, 따라서 더 CECs 사용할 수있다. 우리의 연구에서, 우리는 지역 도살장에서 세포핵이 제거 된 소의 눈을 인수했다. 때문에 Fres의의<html> 소 눈의 시간 자연, 수확 소 CECs의 정확한 수는 눈 당 약 1 × 10 5 세포에서 변동 될 수 있습니다. 반대로 정상적인 인간 각막 CECs 수 안구 당 약 3 × 105이다. 이 숫자는 연구 수준의 각막에 낮과 이식 후 잔여 각막 림에 더 낮은 것입니다. 채취 절차 동안 조작 더 얻어진 세포의 수를 감소시킬 것이다. 또한, 소 CECs이 증식 할 수 있고 자발적으로 EnMT를 받아야; 따라서, 체외 배양 모델에서 소 CEC는 EnMT 메커니즘을 조사 및 타틴 같은 EnMT 억제 화학 물질을 스크리닝에 유용하다. 이러한 CECs의 증식 능력 종의 관련 차이에 대한 우려가 남아 있지만, 우리의 결과는 소 CECs의 EnMT 신호 경로가의 Wnt / β-catenin이 18, 19의 활성화를 포함하여 인간의 CECs의 것과 유사하다는 것을 보여 주었다. 소 CECs의 분리 동안 텐트 &quot;&gt;는 데스 메 막을 박리하는 것이 중요하다. 일부 소 눈에서 데스 메 막을 박리 데스 메막에 과도한 간질 조직을 선도, 각막 기질과 밀착성을 가질 수있다. 또한 트립신이 될 수 있습니다 세포의 순도에 영향을주고, 따라서 실험 결과. 본 연구에서 우리는 현미경 신중 데스 메 막 박리 각막 섬유 아세포로 변환. 상기 세포의 순도를 보장하기 위해 각막 실질 세포의 박리는, 먼저 6에서 수확 된 세포를 배양 세포 합류 할 때까지 cm 접시. 세포 모양의 육각형했다 만 그들은 더 실험에 사용 하였다. 또한 체외 배양 모델에서 소 CEC의 연구 결과를 입증하기 위해, 우리는 이전의 연구 (20, 21)에서 채택 된 쥐의 각막 내피 세포의 cryoinjury 모델을 사용했다. cryoinjury 후, 쥐 CECs은 재생, manife 동안 EnMT을 시행이 Wnt / β-catenin이 신호의 활성화는 CEC 재생하는 동안 종 가운데 보존되어있는 것을 나타내는 활성 β-catenin이의 핵 전좌에 의해 STED. 낮은 CEC 기능은 분명 각막 부종을 초래하기 때문에 또한,이 모델은 CECs의 기능을 조사하는 데 유용합니다. 이러한 초음파 생체 현미경, 전 안부 빛 간섭 단층 촬영 및 공 초점 현미경 등의 장비와 결합, 각막 두께를 정량적으로 평가하여 CEC 기능을 반영 할 수있다. CECs의 재생을 자극하고 상처 치유 후 EnMT을 억제하기 위해, 우리는 타틴 주입 다음의 bFGF의 전방 내 주사를 투여. 치료는 크게 각막 부종의 정도를 감소시켰다. 이전의 연구는 노치 억제제 (21, 22) 등 ROCK 저해제 및 EnMT 억제 제제로서 CEC 성장을 자극 에이전트의 국소 응용 프로그램을 사용하고 있습니다. 그러나, CEC에 약물의 침투 누워ER은 치료 효능 (23)에 영향을 미칠 수있는 각막 상피 세포 및 기질에 의해 제한된다. 국소 도포는 달리, 전방 내 주사는 직접 전방 내로 약물을 도입하여 각막 내피 세포에 직접 액세스 할 수있다. 따라서, 화학 물질의 성장을 자극하고 EnMT이 억제 효과는 다음 cryoinjury unbiasedly 평가 될 수있다. 우리의 연구에서는 전방 내 주입 전 안압을 낮추기 위해 전방 천자를 시행 하였다. 천자없이, 안압의 급격한 증가는 각막 부종 또는 바늘 관을 통해 홍채라도 탈출시​​킨다. 부드럽게 상처를 밀봉하고 안구 감염을 예방 바늘 철수 보조 동안 기관을 압축.

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table><tbody><tr><td>trypsin</td><td>ThermoFisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium and Ham&#039;s F12 medium</td><td>ThermoFisher Scientific</td><td>11330</td><td></td></tr><tr><td>fetal bovine serum</td><td>ThermoFisher Scientific</td><td>26140-079</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>human epidermal growth factor</td><td>ThermoFisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>insulin, transferrin, selenium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>cholera toxin</td><td>Sigma</td><td>C8052-1MG</td><td></td></tr><tr><td>gentamicin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>amphotericin B</td><td>ThermoFisher Scientific</td><td>15290-026</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>111219</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787&amp;nbsp;</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>marimastat</td><td>Sigma</td><td>M2699-25MG</td><td></td></tr><tr><td>anti-active beta-catenin antibody</td><td>Millpore</td><td>05-665</td><td></td></tr><tr><td>anti-snail antibody</td><td>Santa cruz</td><td>sc28199</td><td></td></tr><tr><td>anti-slug antibody</td><td>Santa cruz</td><td>sc15391</td><td></td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>for staining of ABC of bovine CECs</td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11003</td><td>for staining of ABC of rat corneal endothelium</td></tr><tr><td>goat anti-rabbit IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11008</td><td>for staining of snail and slug of bovine CECs</td></tr><tr><td>antibody diluent</td><td>Genemed Biotechnologies</td><td>10-0001</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>laser scanning confocal microscope</td><td>ZEISS</td><td>LSM510</td><td></td></tr><tr><td>xylazine&amp;nbsp;</td><td>Bayer</td><td>N/A</td><td></td></tr><tr><td>tiletamine plus zolazepam</td><td>Virbac</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>proparacaine hydrochloride ophthalmic solution</td><td>Alcon</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>0.1% atropine</td><td>Wu-Fu Laboratories Co., Ltd</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>0.3% gentamicin sulfate</td><td>Sinphar Group</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>basic fibroblast growth factor</td><td>ThermoFisher Scientific</td><td>PHG0024</td><td>clinical drug&amp;nbsp;</td></tr></tbody></table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

소 CECs의 분리 후, 세포를 시험관 내에서 배양 하였다. (1)가 소 CECs의 위상차 이미지를 보여주고있다. 합류 셀의 육각형은 세포가 세포 분리 동안 각막의 간질 섬유 아세포에 의해 오염되지 않은 것으로 나타났다. (2)는 지정된 시점에서 ABC, 달팽이와 민달팽이에 대한 항체를 이용하여 수행 한 면역 염색을 보여주고있다. 외에도 체외 배양 표현형 변화에서, ABC 및 EMT 레귤레이터 대응 핵 전위가 관찰되었다.도 3은 시험 관내 배양 소 CECs의 EnMT 프로세스에 타틴 효과, 광범위한 스펙트럼 MMP 억제제를 나타낸다.도 도 4는 전방 내 주사 하였다 cryoinjury 후 쥐의 외부 눈 사진을 포함한다.도 5는, R의 면역 염색을 나타낸다<html> 각막 버튼에 그 전방 내 주입 한 다음 cryoinjury 후 ABC에 대해 항체를 사용 하였다. ABC의 핵 전좌는 PBS 그룹에서 관찰되었으며 크게의 Wnt / β 카테닌 시그널링 및 EnMT 과정의 활성화를 나타내는 bFGF의 군에서 증가 하였다. 의 bFGF의 전방 내 주입 타틴 주입 한 다음 후, ABC의 핵 염색 타틴의 EnMT 억제 효과를 시사 감소했다. <img alt="그림 1" src="/files/ftp_upload/54329/54329fig1.jpg" /> 그림 1 :. 체외 배양 소 CECs이 배양 접시에 접종 한 후, 소 CECs가 처음 등장의 위상 콘트라스트 이미지가 섬유 아세포와 같은 일 3 6. 그들은 = 하루 9 스케일 바의 완전한 합류에 도달하면 더 육각되었다 50 μm의. 3 복제의 대표 이미지.4329fig1large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 2" src="/files/ftp_upload/54329/54329fig2.jpg" /> 그림 2 :. 활성 β-catenin이 : 체외 배양 소 CECs 소 CECs, ABC, 달팽이와 민달팽이의 핵 전위의 체외 배양 동안 하루 14 ABC를 통해 발견 된의 면역 염색. 스케일 바 = 100 μm의. 3 복제의 대표 이미지. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54329/54329fig3.jpg" /> 그림 3 :. 또는 다른 세포 합류 수준에서 타틴없이 체외 배양 소 CECs의 면역 염색 면역 염색 악마ABC는 달팽이와 민달팽이는 또는 그러나 일 3. 타틴 10 μM없이 소 CECs의 핵에 분명 있다고 보이는 것, 타틴은 크게 9 일 소 CECs의 핵 ABC, 달팽이의 염색 및 슬러그 감소 완전히 합류했다. 스케일 바 = 100 μm의. 3 복제의 대표 이미지. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/54329/54329fig4.jpg" /> 그림 4 :. cryoinjury 후 지정된 시점에서 쥐의 외부 눈 사진, 3 일 연속 cryoinjury 다음은, 쥐가 0.02 ml로, 하루 6 일 일 3. NG / ㎖의 bFGF PBS의 0.02 ml의 50의 전방 내 주입 하였다 PBS가 다른 2 GROU 주입 된 반면, 10 μM 타틴는 bFGF에 / 마리 그룹에 각막 주입PS (N = 9 각 그룹에서). 타틴 더 감소 각막 부종 혼자의 bFGF와 비교. N = 각 그룹의 9 반면 외부의 눈 사진은, bFGF를 주입 한 후 각막 부종을 감소 밝혔다. 스케일 바 = 1mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 5" src="/files/ftp_upload/54329/54329fig5.jpg" /> 그림 5. 9 일에 수확 된 쥐의 각막 버튼의 쥐 각막 버튼의 면역 염색 면역 염색은 PBS 그룹에서 ABC의 작은 핵 염색을 밝혔다. bFGF를 그룹에서 크게 bFGF를 / 마리 그룹에서 감소 된 ABC의 광범위한 핵 염색이 있었다. 스케일 바 = 100 μm의. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54329/54329fig5large.jpg" target="_blank">생의 더 큰 버전을 보려면 여기를 클릭하십시오</a>의 그림입니다.

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

소 각막 내피 세포의 일차 배양 각막 내피 간엽 전이의 메커니즘을 조사 하였다. 또한, 래트 각막 내피 cryoinjury 모델 생체 내 각막 내피 간엽 전환을 증명 하였다.

생체 외 및 생체 내 모델에서 각막 내피 세포 - 중간 엽 전이를 공부하기

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

in-vitro-vivo-models-to-study-corneal-endothelial-mesenchymal

Nanyang Technological University

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JoVE Journal

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

7.9K 조회수.  Far Eastern Memorial Hospital.  소 각막 내피 세포의 일차 배양 각막 내피 간엽 전이의 메커니즘을 조사 하였다. 또한, 래트 각막 내피 cryoinjury 모델 생체 내 각막 내피 간엽 전환을 증명 하였다. 

비디오: In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

각 막 조직 공학:는 생체 외에서 의 모델 인간 각 막의 실질 신경 상호 작용

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

연구

발생학

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

쥐의 각막 이식 : 고체 장기 이식의 가장 일반적인 형태를 연구하기위한 모델

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

의학

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

각막 Micropocket 분석 : 마우스 눈에있는 혈관의 모델

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

신경과학

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

생체 재료 멤브레인에 인간과 양 각막 내피 세포 층의 성장

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

생체공학

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex Vivo 막 기관 문화 모델에 대 한 상처 치유 연구

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

분할 각막 단추를 사용하여 돼지 각막 내피 내피 기관 문화 모형

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

각막 내피 세포 손실의 비 침습적, 레이저 보조 실험 모델의 개발

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

각막 상피의 기계적 및 화학적 손상에 대한 Ex Vivo 및 In Vivo 동물 모델

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

내 피 엽 전환 성장 인자-β 신호 변환에 의해 유도 된의 분자 분석

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

생물학

내피에서 중간엽으로의 전환을 연구하기 위한 생체 내 심장 이식

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

저자 스포트라이트: 내피에서 중간엽으로의 전환 연구를 위한 신생아 이종성 쥐 심장 이식 모델  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

내피에서 중간엽으로의 전환 연구를 위한 신생아 이종성 쥐 심장 이식 모델

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...